PCR is a molecular amplification method routinely practiced in medical and bioresearch settings for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and other types of nucleic acid analysis. For a review of the PCR methodology, see, e.g., PCR Protocols (Methods in Molecular Biology) by Barlett and Stirling (eds.), Humana Press (2003); and PCR by McPherson and Moller, Taylor & Francis (2006).
Digital PCR is a technique that allows amplification of a single DNA template from a minimally diluted sample, thus, generating amplicons that are exclusively derived from one template and can be detected with different fluorophores or sequencing to discriminate different alleles (e.g., wild type vs. mutant or paternal vs. maternal alleles). For a review of the digital PCR methodology, see, e.g., Pohl et al., Expert Rev. Mol. Diagn., 4(1):41-7 (2004). The basic premise of the technique is to divide a large sample into a number of smaller subvolumes (segmented volumes), whereby the subvolumes contain on average a single copy of a target. Then, by counting the number of positives in the subvolumes, one may deduce the starting copy number of the target in the starting volume. Most commonly, multiple serial dilutions of a starting sample are used to arrive at the proper concentration in the subvolumes, the volumes of which are typically determined by a given PCR apparatus. This additional step increases the number of samples to be processed. A set of subvolumes may be tested that statistically represents the entire sample to reduce that number. However, under certain conditions, it may be necessary to detect very lowly expressed genes, resulting in a large number of blank segmented volumes and, thus, a large number of subvolumes to be evaluated. While making a sample more concentrated is a possibility in this case, doing so may introduce significant variability and losses (see, e.g., N. Blow, Nature Methods, 4:869-875 (2007). In addition, a more concentrated sample means that more sample is necessary to begin with.
Further considerations suggest that decreasing the volume of the amplification reaction might improve sensitivity for detecting a single molecule. For example, the TaqMan® assay requires near-saturating amounts of PCR amplification product to detect fluorescence. PCR reactions normally saturate at about 1011 product molecules/microliter due, in part, to reannealing of product strands. To reach this concentration of product after 30 cycles in a 10 μl PCR requires at least 103 starting template molecules. If the volume of the PCR were reduced to ˜10 nanoliters, then a single molecule could generate the required product to be detected by the TaqMan® assay. Attempts have been made to miniaturize PCR volumes (for a review, see, e.g., Zhang et al., Nucl. Acids Res., 35(13):4223-4237 (2007)). Nevertheless, as sample volumes decrease, amplification becomes increasingly more prone to biochemical surface absorption problems due to the increasing surface-to-volume ratio, as well as potential other sources of variability.
Therefore, there exists a need for methods and devices for accurately detecting or quantifying target copy numbers, including by means of the digital PCR.